Amyotrophic lateral sclerosis (ALS) is an idiopathic and fatal neurodegenerative disease of the human motor system¹. Previous studies have revealed that alterations of the corpus callosum (CC) are consistent features of ALS² thus suggest a pathopysiology of reduced interhemispheric neural connectivity. With diffusion tensor imaging (DTI) and resting-state functional MRI (rfMRI), the present study directly examined structural and functional alterations in CC by analysis of voxel mirrored homotopic connectivity (VMHC)³ and diffusion tractography. High-resolution structural MRI (MPRAGE, TR/TE/FA/FOV = 1, 900 ms/2.52 ms/9°/256mm, matrix = 256 × 256, thickness = 1.0 mm, no gap, 176 slices, voxel size = 1 × 1 × 1 mm3), DTI (TR/TE = 10,000 ms/92 ms, 64 diffusion directions with b = 1,000 s/mm2 and an additional volume with b = 0 s/mm2, matrix = 128 × 124, FOV = 256 × 248 mm2, 2-mm slice thickness without gap) and rfMRI (EPI-BOLD, 36 axial slices, slice thickness = 3 mm, TR/TE/FA/FOV = 2, 000 ms/30 ms/90°/192 mm, matrix = 64 × 64, voxel size = 3 × 3 × 3 mm3, total volumes = 240) data were acquired with a 3T Siemens Trio scanner from 38 ALS patients (25M13F, Age=48.50 ± 9.37) and 35 sex- and age-matched control subjects (21M14F, Age=48.82 ± 11.27). The macro-structural metrics extracted from high-resolution structural imaging (thickness, area and volume), micro-structural metrics derived from diffusion imaging (FA, AD, RD and MD) of CC subregions according to the Hofer schema⁴ (Fig. 1), the interhemispheric structural connectivity, reflected as the fiber probability index (FPI) and the interhemispheric functional connectivity measure of voxel mirrored homotopic connectivity (VMHC), were computed and compared between groups.None of the morphometric metrics of the CC subregions was statistical significant after controlling the effects of age, sex and total intracranial volume. The microstructural metrics of CC subregions I, II, III and V were significantly reduced in ALS patients after controlling the effects of age and sex using MANCOVA analysis (Table 1). Extensive VMHC decrements, including the precentral gyrus，the postcentral gurus, the paracentral lobule, the superior temple gyrus, the cingulate gyrus, the putamen and the superior parietal lobule, were found in ALS (AlphaSim corrected, P< 0.01)(Fig. 2, Table 2). Only the FPI values through CC subregion III, connecting the bilateral motor cortices, were significantly decreased in the ALS patients compared with the controls (Fig. 3, Table 3). Extensive alterations in the micro-structural metrics in CC subregions in ALS patients. These findings support the notion of interhemispheric disconnection in ALS. In addition, VMHC was applied for the first time to investigate interhemispheric FC in ALS. The patients exhibited lower VMHC in extensive brain areas, including precentral gyrus, paracentral and postcentral gyrus, relative to control subjects. These results indicate that there is extensive disruption of interhemispheric functional connectivity in ALS. In contrast to extensive alterations in interhemispheric functional connectivity, we only revealed the interhemispheric structural connectivity impairment in the CC subregion III in ALS compared to controls. The discrepancy of the alterations between the interhemispheric functional and structural connectivity in ALS maybe indicate that the functional connectivity is more sensitive to interhemispheric connectivity disruption⁵.The findings of functional and structural connectivity reductions in ALS here supports the notation of interhemispheric connectivity impairment in ALS. They also provide an important new avenue to understand the pathophysiological nature of ALS.